Protein kinases play pivotal roles in signal transactions, and dysfunction of them is a key factor for diseases like cancer, inflammation, diabetes etc. Kinome drug discovery is a major focus in pharmaceutical industry, and high throughput screening (HTS) is a major tool for lead discovery. Reaction Biology Corporation's (RBC) Discovery Dot miniaturization technology will not only facilitate the drug discovery process but also save the cost by millions. RBC has developed extremely versatile nanoliter reaction microarrays to serve markets for ultra uHTS drug discovery, large scale IC50 determinations, and large scale IC50 selectivity/toxicity profiling. These reactions are 1000 to 10,000-fold smaller than well plate formats currently used widely in drug discovery. Through the power of contact printing, RBC has the capacity to print and activate over 3 million reactions per week. Currently, 3072 drug screening reactions can be carried out on a single microarray. The ability to print large chemical libraries in hundreds of replicates sets for kinase and phosphatase screening can drive chemical-proteomic research, environmental toxicology, hit discovery and prioritization, lead selection, and ADMET. In preliminary studies, RBC has demonstrated the ability to conduct calibrated screening of human protein kinases (c-src, PKA) using printed microarrays. Through Phase I funding, the proposed research will focus on the following three aims: Aim 1 Validation of 5 tyrosine kinase and 5 serine/threonine kinase assays with Z'>0.6 and further optimization to achieve the lowest cost assays in the industry; Aim 2 Design, implementation, and validation of new assay methods for kinases and phosphatases that are amenable to microarray-based screening of chemical libraries; Aim 3 Validation of kinase and phosphatase HTS on microarrays against well-plate assay using a standardized screening library. This work will enable Phase II activities where Reaction Biology will expand its assay range across the human kinome using a universal kinase assay against large synthetic and natural libraries.